This invention relates to macromolecules containing biologically active drugs/biomolecules, or precursors thereof, and fluoroligomers; compositions comprising said macromolecules containing biologically active drugs/biomolecules and fluoroligomers in admixture with polymers, particularly biomedical polymers; articles made from said admixtures, particularly medical devices; and methods of preparation of said macromolecules containing biologically active drugs/biomolecules and fluoroligomers.
Biomedical polymers (includings polyamides, polyurethanes, polysilicones, polyfluorocarbons, polysulfones, polyolefins, polyesters, polyvinyl derivatives, polypeptide derivatives, polysaccharide derivatives etc. ) are applied extensively in the manufacture of conventional biomedical devices used in contact with living tissues, body fluids and its constituents, such as vascular and skin grafts, endotracheal tubes and catheters, drug delivery vehicles and affinity chromatography systems [1]. Many synthetic polymers have characteristics that make them useful as biomedical materials. One reason for this is the wide range of properties available from man-made polymers. The chemistry of the repeat unit, the shape of the molecular backbone, and the existence and concentration of intermolecular bonds among the macromolecules that make up the polymeric material all influence its ultimate properties. Additional variations in polymer character is possible in polymers with more than one kind of repeating unit. Copolymers, terpolymers, and even multipolymers are possible in which the properties of more than one polymer type are combined to produce a unique material. The arrangement of the different repeat units in copolymers allows further property variations. The overall concentration of each monomer is also an important parameter in determining the properties of the copolymers, but unless one monomer is used in great excess over the other, the resulting properties can be quite different from either homopolymer.
Biocompatibility
Biocompatibility is defined as the ability of a material to perform with an appropriate host response in a specific application. The host relates to the environment in which the biomaterial is placed and will vary from being blood, bone, cartilage, heart, brain, etc. Despite the unique biomedical related benefits that any particular group of polymers may possess, the materials themselves, once incorporated into the biomedical device, may be inherently limited in their performance because of their inability to satisfy all the critical biocompatibility issues associated with the specific application intended. For instance while one material may have certain anti-coagulant features related to platelets it may not address key features of the coagulation cascade, nor be able to resist the colonization of bacteria. Another material may exhibit anti-microbial function but may not be biostable for longterm applications. The incorporation of multi-functional character in a biomedical device is often a biomedical device is often a complicated and costly process which almost always compromises one polymer property or biological function over another, yet all blood and tissue contacting devices can benefit from improved biocompatibility character. Clotting, toxicity, inflammation, infection, immune response in even the simplest devices can result in death or irreversible damage to the patient. Since most blood and tissue material interactions occur at the interface between the biological environment and the medical device, only the make-up of the outer molecular layer (at most the sub-micron layer) of the polymeric material is relevant to the biological interactions at the interface. This means that as long as the polymer does not contain any leachable impurities, the chemistry of the bulk polymer, which is distant from the biological interface, should have a minimal influence on tissue and body fluid interactions if the material surface is relatively biostable.
Surface Modification
Given the knowledge that it is the surface that is the most pertinent issue in the matters of biocompatibility, a practical approach taken towards the development of biomedical devices has involved the utilization of polymeric materials that satisfy the bulk material criteria for the device while applying some form of surface modification which may specifically tailor the biological surface properties and produce minimal change to the bulk character. Such an approach is seen advantages over grafting biologically active agents to the bulk polymer chains since the latter approach brings about significant changes to the physical structure of the polymers [2]. Methods that have been used for the surface modification of polymer surfaces rather than bulk grafting of the polymers have included the following: Non-covalent coatings (with and without solvent), chemical surface grafting, ion implantation, Langmuir-Blodgett Overlayer and self assembled films, surface modifying additives, surface chemical reactions and etching and roughning.
Surface Modifying Macromolecules
The use of oligomeric surface modifying additives present a significant advance over many commercial surface coating technologies reported above (i.e. radiation grafting polymerization; chemical coating, solvent coating; electron radiation; plasma polymerization or deposition; etc.), since it is a one step operation which can be simultaneously carried out with normal extrusion, film casting, fibre spinning and injection molding processes. The technology is readily transferable from one field to the next because it is adaptable to different polymer systems, analogous to additives such as colorants. General applications have included desoiling agents [3] and membrane applications for the separation of organics and water [4]. In areas of specific interest to the biomedical field, polymeric additives have been developed for applications in polyurethanes and other materials [5,6]. Ward et al. [7], issued describes polymer admixtures formed from a base polymer and thermoplastic copolymer additives having polar hard segments and polar and no-polar soft blocks in graft or block copolymer form, for use in biomedical devices. Ward et al. [7], describes novel linear polysiloxane-polylactone block copolymers, particular polysiloxane-polycaprolactone linear blocked copolymers, miscible with nylon for use as surface-modified nylon articles. Ward et al., [8] describes end-group containing polymers that comprise a linear base polymer having covalently bonded surface active end groups of a nature and present in an amount such that the polymer has a surface interaction tension that differs by at least 1 dyne/cm from the surface or interfacial tension of an otherwise identical polymer that does not contain the covalently bonded surface active endgroups. Santerre [6] describes fluoroligomers and compositions comprising fluoroligomers as surface-modifiers in admixture with polymers, for providing articles with passive surface properties, particularly, medical devices that shield enzyme interactions along with having acceptable passive blood compatibility.
It should be noted that in cases pertaining to the end group""s described above [6,8], and the influence of the latter on cells, proteins and other biomolecular functions, the type of the interaction is relatively non-specific and it is preferred to be passive in nature, meaning that the surface generated by the end groups does not contain in itself a defined biochemical action that allows it to be both surface active and express a specific biological action on individual cellular mechanisms, specific protein or enzyme activity, or messenger action in the case of peptide signaling molecules. For the latter, the biomedical community still relies on traditional methods of therapy, i.e. the delivery of drugs or bio-active molecules via traditional diffusion mechanisms. Classically this has been achieved systemically but over the last decade a host of localized delivery vehicles have been developed and consist primarily of diffusion controlled systems or polymeric substrates with surface grafted drugs of bio-active molecules [9-11]. xe2x80x9cFor example, Santerre and Mittleman [9] teaches on the synthesis of polymeric materials using pharmacologically-active agents and monomers for polymers. The pharmacologically-active compounds provide in vivo enhanced long term anti-inflammatory, anti-bacterial, anti-microbial and/or anti-fungal activity.xe2x80x9d
Specifically, polymeric carriers have been developed, which contain drug moieties as terminal groups, or as pendent groups on the polymer chain. Polymers that are utilized for conjugation with drugs have included poly(xcex1-amino acids), polysaccharides such as dextrans and chitin, polyurethanes and others. By copolymerizing amino acid moieties into the backbone of the polymer chains, Nathan [12] et al. have synthesized polyurethanes having pendent drugs to the amino acid unit. The specific conjugation of penicillin V and cephradine as pendant antibiotics to polyurethanes has been reported on by Nathan [12]. In the latter work the investigators showed that hydrolytically labile pendant drugs were cleaved and exhibited antimicrobial activities against S. aureus, E. faecalis and S. pyogenes. Others have described vinyl monomers with nalidixic acid, a quinolone antibiotic, coupled in a pendant manner to the active vinyl molecule, which was subsequently polymerized. In in-vivo hydrolysis studies they reported a 50% release of drug moities over the first 100 hours. This quinolone drug has been shown to be effective against gram negative bacteria in the treatment of urinary track infections, however chemical modifications of the latter (e.g. ciprofloxacin, norfloxacin and others) have a wider spectrum of activity. More recent work on the conjugation of norfloxacin to mannosylated dextran has been carried out in an effort to increase the drug""s uptake by cells, enabling them to gain faster access to micro-organisms [13]. The studies showed that norfloxacin could be released from a drug/polymer conjugate by enzyme media and in in vivo studies, the drug/polymer conjugate was effective against Mycobacterium tuberculosis residing in liver [13]. In the later system, norfloxacin was attached pendent to sequences of amino-acids which permitted its cleavage by the lysosomal enzyme, cathepsin B.
In all drug conjugates, the goal has been to develop systems that would enhance either the drug activity and/or the diffusivity of the drug into an aqueous biological environment. In some instances the conjugates may be loaded into substrates, polymeric or non-polymeric, in order to have the conjugate gradually released from the substrate (i.e. controlled drug delivery). In other instances the drug may be actually grafted to the polymer matrix chains, either to remain permanently fixed or subsequently released via hydrolysis of the coupling bond. However, the latter limits the drug""s diffusion among the polymer chains making up the matrix, thus limiting the delivery of the drug. It should be noted that in either case, i.e. the drug loading of a polymer matrix or coupling to a polymer matrix, there is the introduction of a significant effect on bulk polymer properties because the drug is distributed throughout the material. In the particular application of the latter systems, where drug delivery (i.e. biochemical function) is the key focus, and the physical function of the device rather than the biochemical function, may be secondary or have no function over the longterm (i.e. weeks to years), consideration to the changes in bulk structure are not a limitation of the device""s primary function. However, such systems would not readily satisfy the physical demands of most implant devices (i.e. heart valves, vascular grafts, catheters, corneal lens, tendons, tissue scaffolds etc.) because they would compromise physical function which is important to the device""s role. A drug conjugate applied to such system would however have a significant advantage if it could be surface specific rather than bulk distributed. Furthermore, if such a drug conjugate could achieve surface specificity without compromising the biochemical potential of the drug component, then one would have a means of generating simultaneous surface modification and introduction of specific biochemical function to the surface of an implant device (applied in body part replacement) or a structural scaffold (applied in tissue engineering or bio-reactor systems). This would provide the field with a truly unique technology which could rival existing systems since at the current time, there are few if any technologies related to biomaterials and their associated devices (i.e. vacular grafts, tendons, corneal lens, and the like), which simultaneously provide for a specific and stable surface modification of the biomaterial, by a drug or drug/conjugate loaded component.
Publications
(1) Ratner B. D., Hoffman, A. S, Schoen F. J., Lemons, J. E., Biomaterials Science, xe2x80x9cAn Introduction to Materials in Medicinexe2x80x9d, Academic Press, San Diego, 1996.
(2) Santerre, J. P., Brash, J. L., J. Appl. Polym. Sci., 51, 515 (1994).
(3) Eur. Patent. Appl. 0,231,927, Submitted by Asahi Glass Company Ltd., Mar. 2, 1987.
(4) U.S. Pat. No. 5,954,966xe2x80x94Matsuura et al., issued Sep. 21, 1999.
(5) U.S. Pat. No. 4,861,830xe2x80x94Ward, Robert S., issued Aug. 29, 1989.
(6) U.S. Pat. No. 6,127,507xe2x80x94Santerre, Paul J. Oct. 3, 2000.
(7) U.S. Pat. No. 5,235,003xe2x80x94Ward, Robert S. Aug 10, 1993.
(8) U.S. Pat. No. 5,589,563xe2x80x94Ward, Robert R. and White, Kathleen A. Dec. 31, 1996.
(9) U.S. Pat. No. 5,798,115xe2x80x94Santerre, Paul J. and Mittleman, Marc W. Aug. 25, 1998.
(10) Modak S. M., Sampath, L., Fox, C. L., Benvenisty A., Nowygrod, R., Reemstmau, K. Surgery, Gynecology and Obstertrics ,164, 143-147 (1987).
(11) Bach, A.; Schmidt, H.; Bxc3x6ttiger, B.; Schreiber B.; Bxc3x6hrer, H.; Motsch, J.; Martin, E.; Sonntag, H. G., J. Antimicrob. Chemother., 37, 315, (1996).
(12) Nathan, A.; Zalipsky, S.; Ertel, S. I.; Agarthos, S. N.; Yarmush, M. L.; Kohn. J. Bioconjugate Chem. 1993, 4, 54-62.
(13) Roseeuw, E.; Coessens V.; Schacht E., Vrooman B.; Domurado, D.; Marchal G. J Mater. Sci: Mater. Med 1999, 10, 743-746.
It is an object of the present invention to provide polymer compounds comprising biologically active molecules, such as, for example pharmaceuticals including anti-inflammatory, anti-oxidant, anti-coagulant, anti-microbial, cell receptor ligands and bio-adhesive molecules, oligonucleic acid sequences for DNA and gene sequence bonding, phospholipid head groups to provide cell membrane mimics or precursors thereof, and fluoroligomers.
It is a further object of the present invention to provide said polymer compounds in admixture with a compatible polymeric biomaterial or polymer composite biomaterial for providing a shaped article having improved surface properties.
It is a further object of the present invention to provide said shaped article for use as a medical device, comprising a body fluid and tissue contacting device in the biomedical sector, or in providing improved biocompatibility, or for use in the biotechnology sector for improving affinitity column chromatography systems or promoting surface catalytic reactions.
It is a further object of the present invention to provide said polymer compounds in admixture with either a base polyurethane, polysilicone, polyester, polyethersulfone, polycarbonate, polyolefin or polyamide for use as said medical devices in the biomedical sector, or in providing improved biocompatibility, or for use in the biotechnology sector for improving affinitity column chromatography systems, design diagnostics and biosensor chips or promoting surface catalytic reactions.
It is a further object of the invention to provide processes of manufacture of bioactive fluoroalkyl surface modifiers, said polymer compounds, said admixtures and said shaped articles.
The invention, generally, provides a bioactive fluoroalkyl surface modifier, herein termed a BFSM, having a central portion comprising oligomeric segments of  less than 15,000 theoretical molecular weight and optional link segments, herein denoted [linka] covalently coupled to a first oligomeric segment denoted [oligo], such that the central portion is compatible with the polymeric material in which the BFSM is subsequently used in admixture, xcex1-xcfx89 terminal polyfluoro oligomeric groups denoted [fluoro], non-optional coupled link segments herein denoted [linkB], wherein [linkB] is covalently coupled with the central portion and [fluoro] as well as being covalently or ionically coupled to a bioactive component.
Accordingly, the invention provides in one aspect, a bioactive fluoroalkyl surface modifier for use in admixture with a compatible base polymer, said modifier having the general formula 
is a central portion comprising an oligomeric polymeric segment having a theoretical molecular weight of less than 15,000, and being compatible with said base polymer; wherein
[oligo] is a first oligomeric segment;
[link A] is a second coupling segment linking one [oligo] to another [oligo] within said central portion;
n is 0 to 20;
[fluoro] is a polyfluoro oligomeric group; and
[link B] is a first coupling segment linking said central portion to said [fluoro] through said first coupling segment; and coupled to a bioactive moiety [Bio] or precursor thereof; and
m is 1 to 20.
In a preferred aspect the invention provides a modifier as hereinabove defined of the general formula 
wherein
n is 0-20; and
m is 1-20; provided that when n is 0, m is1.
In a further preferred aspect the invention provides a bioactive fluoroalkyl surface modifier for use in admixture with a compatible base polymer, said modifier having the general formula 
wherein [[oligo]-(link A-[oligo])n] is a central portion comprising an oligomeric polymeric segment having a theoretical molecular weight of less than 15,000, and being compatible with said base polymer;
[oligo] is a first oligomeric segment;
[link A] is a second coupling segment linking one [oligo] to another [oligo] within said central portion;
n is 0 to 20;
[fluoro] is a polyfluoro oligomeric group; and
[link B] is a first coupling segment linking said central portion to said [fluoro] through said first coupling segment; and coupled to a bioactive moiety [Bio] or precursor thereof; and
m is 1 to 20.
Preferably, n is 2 to 10 and m is 1 to 10.
It can be seen in the above formula that [link B] is both within and outside of said central portion.
By the term xe2x80x9coligomeric segmentxe2x80x9d is meant a relatively short length of a repeating unit or units, generally less than about 20 monomeric units and molecular weights less than 5000. Preferably, [oligo] is selected from the group consisting of polyurethane, polyurea, polyamides, polyalkylene oxide, polycarbonate, polyester, polylactone, polysilicone, polyethersulfone, polyolefin, polyvinyl, polypeptide polysaccharide; and ether and amine linked segments thereof.
By the term xe2x80x9clinkA moleculexe2x80x9d is meant a molecule capable of covalently coupling oligo units together and to form said second coupling segments within said central portion. Typically, linkA molecules can have molecular weights ranging from 40 to 700 and have difunctionality to permit coupling of two oligo units. Preferably the linkA molecules selected from the group of diamines, diisocyanates, disulfonic acids, dicarboxylic acids, diacid chlorides and dialdehydes. Terminal hydroxyls, amines or carboxylic acids on the oligo molecules can react with diamines to form oligo-amides; react with diisocyanates to form oligo-urethanes, oligo-ureas, oligo-amides; react with disulfonic acids to form oligo-sulfonates, oligo-sulfonamides; react with dicarboxylic acids to form oligo-esters, oligo-amides; react with diacid chlorides to form oligo-esters, oligo-amides; and react with dialdehydes to form oligo-acetal, oligo-imines.
By the term xe2x80x9clinkB moleculexe2x80x9d is meant a molecule capable of providing primary functional groups capable of covalently coupling with the oligo/linkA central portion and fluoro group components, as well as simultaneously having secondary functional chemistry for coupling drug or bioactive components herein termed Bio to constitute said first coupling segment. Typically, linkB molecules have molecular weights ranging from 40 to 700. Preferably the linkB molecules are selected from the group of functionalized diamines, diisocyanates, disulfonic acids, dicarboxylic acids, diacid chlorides and dialdehydes, wherein the functionalized component has secondary functional chemistry that is accessed for chemical attachment of [Bio] components. Such secondary groups include, for example, esters, carboxylic acid salts, sulfonic acid salts, phosphonic acid salts, thiols, vinyls and secondary amines. Again, terminal hydroxyls, amines or carboxylic acids on the oligo/linkA intermediates can react with diamines to form oligo-amides; react with diisocyanates to form oligo-urethanes, oligo-ureas, oligo-amides; react with disulfonic acids to form oligo-sulfonates, oligo-sulfonamides; react with dicarboxylic acids to form oligo-esters, oligo-amides; react with diacid chlorides to form oligo-esters, oligo-amides; and react with dialdehydes to form oligo-acetal, oligo-imines.
Typically, the [fluoro] polyfluoro oligomeric group has a molecular weight ranging from 100 to 1500, and generally formed in the BFSM by reaction of the corresponding perfluoroalkyl group, having precursor monofunctional hydroxyl or amine groups, with the link B molecule.
Preferably, [fluoro] is selected from the group consisting of radicals of the general formula CF3(CF2)pCH2CH2xe2x80x94 wherein p is 2-20 and CF3(CF2)m(CH2CH2O)q-wherein q is 1-10 and m is 1-20. More preferably [fluoro] is the perfluoroalkyl group C8F17CH2CH2xe2x80x94
By the term xe2x80x9cdrug or biologically active agentxe2x80x9d, or precursor thereof, is meant a molecule that can be coupled to linkB segment either via covalent or ionic bonding. The molecule must have some specific and intended pharmaceutical or biological action. Typically the [Bio] unit has a molecular weight ranging from 40 to 5000 but may be higher if it does not inhibit transport of the BFSM to the surface of the material being used to form the intended shaped articles. Preferably, the Bio unit is selected from the group of anti-inflammatory, anti-oxidant, anti-coagulant, anti-microbial, cell receptor ligands and bio-adhesive molecules, specifically oligo-peptides and oligo-saccharides, oligonucleic acid sequences for DNA and gene sequences bonding, and phospholipid head groups to provide cell membrane mimics. The Bio component must have at least one chemical function that can react with the secondary groups of the linkB component.
The oligomeric polymeric segment preferably has a molecular weight of  less than 10,000; and more preferably,  less than 5,000.
The term xe2x80x9ctheoretical molecular weightxe2x80x9d in this specification is the term given to the absolute molecular weight that would result from the reaction of the reagents utililized to synthesize any given BFSM. A close confirmation of this absolute value can be ascertained by elemental analysis of the fluorine content which can be correlated to the final absolute molecular weight of the polymer. As is well known in the art, the actual measurement of the absolute molecular weight is complicated by physical limitations in the molecular weight analysis of polymers using gel permeation chromatography methods. Hence, in some instances, a polystyrene equivalent molecular weight is reported for gel permeation chromatography measurements. The latter number is merely of value in terms of reporting on reproducibility of the molecular weight for a given BFSM. It is the theoretical molecule weight (i.e. absolute molecular weight based on reagent stochiometry) which is of relevance in defining the limitations herein, since the latter defines the fluorine content. The fluorine content of the BFSM, preferably, should remain above 1 wt % in order to enable the molecule to effectively migrate to the polymer surface in admixture applications.
In a further aspect, the invention provides compositions of a base polymer in admixture with a bioactive fluoroalkyl surface modifier (BFSM), as hereinabove defined, preferably in the form of a shaped article.
Examples of typical base polymers of use in admixture with aforesaid BFSM according to the invention, includes polyurethanes, polysulfones, polycarbonates, polyesters, polyethylene, polypropylene, polystyrene, polysilicone, poly(acrylonitrile-butadienestyrene), polybutadiene, polyisoprene, polymethacrylate, polyvinylacetate, polyacrylonitrile, polyvinyl cloride, polyethylene terephtahate, cellulose and other polysacharides. Preferered polymers include polyamides, polyurethanes, polysilicones, polysulfones, polyolefins, polyesters, polyvinyl derivatives, polypeptide derivatives and polysaccharide derivatives.
The admixed compositions according to the invention may be used as a surface covering for an article, or, most preferably, where the composition comprises a base polymer of a type capable of being formed into 1) a self-supporting structural body, 2) a film; or 3) a fiber, preferably woven or knit. The composition may comprise a surface or in whole or in part of the article, preferably, a biomedical device or component thereof; an affinity column for pharmaceutical or biomolecule purification, or microfilm form for diagnostic and bio-sensor applications.
In a preferred aspect, the invention provides an admixed composition, as hereinabove defined, comprising in admixture either a polyurethane, polysilicone, polyester, polycarbonate polysaccharide with a compatible BFSM, in a surface modifying enhancing amount of preferably 0.5-10 w/w %, more preferably 1-5 w/w %, more preferably 2-10 w/w % of the resultant admixed composition. In the case of a polyurethane base, it should have a molecular weight of at least 1.05 times the molecular weight of the BFSM.
Thus, this invention, in one aspect, defines a family of novel bioactive fluoroalkyl surface modifiers that have fluorinated tails at each end of the molecule and bioactive molecules grafted to [linkB] segments within the chain of the surface modifier. The centre of the BFSM is tailored to be compatible with the base polymer substrate to which it is added.
The BFSMs, according to the invention, are synthesized in a manner that they contain a base polymer compatible segment, terminal hydrophobic fluorine components which are non-compatible with the base polymer and a bioactive moiety containing biochemical function with either inherent anti-coagulant, anti-inflammatory, anti-oxidant, anti-microbial potential, cell receptor ligands, e.g. peptide ligands and bio-adhesive molecules, e.g. oligosaccharides, oligonucleic acid sequences for DNA and gene sequence bonding, phospholipid head groups to provide cell membrane mimics, or a precursor of the bioactive moiety.
The base polymer compatible segment of the BFSM i.e. the [oligo] [linkA] and [linkB] segments is selected to provide an anchor for the BFSM within the base polymer substrate upon admixture. While not being bound by theory, it is believed that the oligomeric fluorine tails, which are not miscible with the base polymer, provide a significant driving force for carrying the the BFSM towards the surface, with the terminal ends of the BFSM oriented outwards of the surface. The latter process is believed to be driven by the thermodynamic incompatibility of the fluorine tails with the base polymer substrate, as well as the tendency towards establishing a low surface energy at the mixture""s surface. When the balance between anchoring and surface migration is achieved, the BFSM remains stable at the surface of the polymer, while simultaneously altering surface properties. Since the biologically active compound is coupled immediately adjacent to the xcex1-xcfx89 fluorine tails of the BFSM in the linkB segment, they will also be preferentially delivered to the surface of the base polymer substrate.
I have found that the utility of the additives of the invention versus other known macromolecular additives or drug polymer conjugates, lies in:
1) BFSMs are relatively low molecular weight compounds of  less than 15,000, which allows them to more readily diffuse among the macromolecular polymer chains of the base material;
2) BFSM can modify surfaces at less than 5 wt % of the BFSM relative to the weight of the base polymer to which they are added. This is an important attribute because it minimizes significant bulk changes to the base materials to which BFSM is added, and therefore, allows for a specific biological activity to be present at the surface, while the bulk base material performs its intended task. For example, if a 3-dimentional porous matrix is desired for a tissue engineered structure with low-swelling function, such a material could not be constructed of traditional biogel polysaccharide matrices. However oligomeric saccharides with cell adhesive character may be synthesized into a BFSM for delivery to the surface of a low swelling material such as a L-poly-lactic acid. In this system, surface bio-activity can be tailored separately from bio-resorbing rates for the base polymer. This is often desired in the case where mechanical function is desired to remain relatively stable over the tissue integration period.
3) BFSM simultaneously establishes a fluorocarbon segment at the surface and a biological or pharmaceutical agent pendent adjacent to the fluorocarbon segment chemistry. The fluorocarbon base provides in terms of surface energy, a relatively neutral surface, which does not promote strong cell adhesion and minimizes protein activation. This feature is strategic since relative to the remainder of the surface, it permits the [Bio] component adjacent to the fluorocarbon base to have a specific cellular or biomolecular interaction with the intended target i.e. for example, platelet, bacteria, thrombin, and the like. The surface modification achieved by the simultaneous combination of surface passivation i.e. xcex1-xcfx89 fluorine tails and target biological function has not been previously achieved and/or demonstrated with any other family of surface amphipathic polymeric type surface modifying macromolecules or drug polymer conjugate.
4) The [linkB] molecule contains one or several functional groups for attachment of the [Bio] component. Depending on the nature of the [linkB] functional group(s), the [Bio] component may be rendered stable for local surface function or hydrolysable for the delivery of Bio components remote from the polymeric implant surface. The introduction of such capabilities has not been previously achieved and/or demonstrated with any other family of surface amphipathic polymeric type surface modifying macromolecules.
5) All BFSMs use similar fluorocarbon oligomers to drive the BFSM to the surface. This permits the delivery of different types of [Bio] components to the surface by simply addition of a blend of different BFSM additives to the desired polymer matrix. For example, it may be desired for a blood contact material to deliver an antibiotic, anticoagulant and a peptide ligand to the surface of a polymer. The former would provide an acute defense against initial bacterial challenges, the anti-coagulant could control acute thrombogenenic events and the peptide ligand may provide a longterm binding site for re-endothelializing a surface. This represents a controlled multi-functional surface modification not previously achieved and/or demonstrated with any other surface modification technology in the biomedical and biotechnological discipline.
6) Since a BFSM has the potential to migrate to the surface during processing i.e. film forming, extrusion, fibre forming, and the like, the present invention provides for the elimination of post-processing steps for introducing bio-active molecules at the surface, as is required with other techniques in the field i.e. radiation grafting polymerization; chemical coating, solvent coating; electron radiation; plasma polymerization or deposition; and the like. The provision for such capabilities represents surface modification approach not previously achieved and/or demonstrated with any other surface modification technology in the biomedical and biotechnological discipline.
The surface modifying agents according to the invention significantly alter the surface chemistry and biochemistry of, for example, segmented polyurethanes, i.e. for example, a BFSM containing a natural anti-oxidant e.g. vitamin-E can migrate to the surface of the polymer mixture and exhibit a new hydrophobic surface. The advancing contact angle, which is a measure of the surface""s hydrophobic components for the examples, hereinafter described, shows significant increases and parallel values with those of typical fluoropolymers i.e. 116xc2x0 for the advancing contact angle of Teflon(copyright) fluorocarbon. The advancing contact angle measurement therefore becomes an effective tool for assessing the extent of change introduced when the fluorine segments of the BFSM direct the molecule to the surface. A further confirmation of the specific affinity of the BFSM at the surface is to assess elemental change, with fluorine being an easy marker. X-ray photo-electron spectroscopy is an effective tool for identifying changes in elemental types and distributions within the upper 10 nm of the surface. For the polyurethane examples herein described, it is found that a 5 wt % of the BFSM relative to the polymer can have an atomic percentage of fluorine in excess of 40%, whereas the background fluorine values for the non-modified polyurethane is less than the detection limits of 1-2 atomic %.
The presence of the drug, adjacent to the [fluoro] segment of the BFSM at the surface, can also be assessed by changes in advancing contact angles, as well as by changes in the specific bio-activity at the surface. The introduction of a typical organic, i.e. carbon/oxygen/nitrogen/hydrogen atom-containing based drug adjacent to the surface fluorine, reduces the advancing contact angle of the surface, relative to that of a surface with surface modifiers only containing the terminal fluorine groups. Specific activity can be assessed based on the units of measure for a particular molecules bio-function. For instance, for a BFSM containing an anti-coagulant, such as heparin, the activity can be measured by determining the deactivation of thrombin in the presence anti-thrombin, while the activity of vitamin-E can be measure by the surfaces ability to quench free radicals generated by oxidants.
The BFSM""s are, for example, of use with, but not limited to, linear or crosslinked polysilicone, polyester, polyurethane, polyethersulfone, polycarbonate, polyolefin and polyamide materials. By tailoring the central portion of the BFSM, the present invention may be applied, inter alia, to a wide range of polymer materials, which include polymers synthesized with reagents that are of common knowledge in the field of segment polyurethanes. This class of polymers is composed of heterogeneous compounds in which, quite often, the urethane groups themselves only make up a fraction of the dominant functional linkages within the macromolecular chains. These include, but are not limited to, various diisocyanates, oligomeric precursor components and low molecular weight chain extender components.
There are no restrictions on the specific stoichiometry of the reagents used in the synthesis of the BFSM. With the exception of the [Bio] components, there is no restriction in the manner in which the reagents are added to each other, the temperature, pressure or atmosphere under which they are synthesized or the use of catalysts in their reaction. However, [oligo] components are of relatively short length in terms of the repeating unit or units, and are generally less than about 20 monomeric units and of molecular weights less than 5000. Typically, linkA molecules have molecular weights ranging from 40 to 700 and must have difunctionality to permit coupling of two [oligo] units. Typically, [fluoro] units can have molecular weights ranging from 100 to 1500; and linkB molecules have molecular weights ranging from 40 to 700. [Bio] units have molecular weights ranging from 40 to 5000, but may be higher if they do not inhibit transport of the BFSM to the surface of the material being used to form an intended shaped article. It is not desirable to simultaneously synthesize a BFSM additive with the base polymer is which they are admixed, since the synthesis of the BFSM additive may be sensitive to reaction conditions of other polymers. As well, it is not desirable to carry out the Bio component coupling at the same time as reacting the other reagents in the production of the BFSM, since the Bio compiling reaction may be sensitive to reaction conditions. The BFSM as an additive may be added to a base polymer synthesis reaction, in such a manner as to incorporate the BFSM additive into the base polymer substrate, prior to the final work-up of the polymer substrate.
In order to illustrate the design of BFSM additives for common polymers as the base polymer, and to describe the rationale for the selection of the BFSM candidates, five polymers were used as representative compounds compatible with the list of reagents given, hereinbefore. One material is MED10-6640 silicone dispersion Pt catalyst, polydimethylsiloxane elastomer from Nusil Silicone Technology. Another is a polycarbonate based polyurethane (HDI/PCN/BD) synthesized from 1,6 hexamethylene diisocyanate, polycarbonate of molecular weight 970, butane diol and dibutyltin dilaurate catalyst. The remaining three polymers were polyethersulfone (PES 4100P(trademark)) from ICI chemicals, polypropylene and nylon 6,6 (Aldrich). Thus, the reagents and stoichiometry used in the synthesis of the BFSM according to the invention for these particular materials favour chemical compatibility with the base, i.e. have an appropriate arrangement of polar versus non-polar character. Clearly, a BFSM based on a polyamide [oligo] component would not be chemically compatible with a polysiloxane base elastomer. A balance between chemical compatibility with the base polymer and specific migration towards the surface of the base polymer is preferably achieved by keeping the molecular weight of the BFSM between the ranges of 500 to 15,000. If the central component of the BFSM, made up of [oligo], [linkA] and [link B] segments, is too large, for example, typically  greater than 30 times that of the [fluoro] segments, it is difficult for the surface driving terminal [fluoro] segment to effectively attain residence at the surface of the base polymer during the time of device processing. Stronger interactions resulting from dispersion and dipole/dipole forces between the BFSM""s central portion, composed of [oligo], [linkA] and [link B] segments, and the base polymer results in overall lower molecular weights for the BFSM. The latter also favor surface migration of the BFSM. Therefore, control of the molecular weight in the synthesis of an effective BFSM is highly desirable in its ability to modify the surface chemistry of a polymer substrate.
The BFSM may be synthesized using a multi-functional linkA molecule, a multi-functional oligo molecule, a multi-functional linkB molecule, a molecule and a Bio molecule having at least a functional component that can be covalently coupled to the BFSM via the secondary function of the [linkB] segment. The linkA and the primary function of the linkB molecules are preferably, but not so limited, to be di-functional in nature, in order to favour the formation of a linear BFSM. Linear, as apposed to branched or crosslinked BFSMs, have better migration properties within the base polymers, since interactions resulting from dispersion and dipole/dipole forces are reduced. Preferred linkA molecules for biomedical and biotechnology applications are diisocyanates: for example, 2,4 toluene diisocyanate; 2,6 toluene diisocyanate: methylene bis (p-phenyl) diisocyanate; lysine diisocyanato esters; 1,6 hexane diisocyanate; 1,12 dodecane diisocyanate; bis methylene di (cyclohexyl isocyanate); trimethyl- 1,6 diisocyanatohexane. The molecular weights of the [oligo] groups are between 200 to 5000, but preferably have molecular weights of less than 3500. Synthesis of the central portion of the BFSM can be carried out by classical reactions using the desired combination of reagents.
In the final step, a Bio component is coupled to the secondary function of the link B segment. These reactions are typically carried out by classical nucleophilic reactions and may involve a pre-activation of the secondary site on the link B component. For example, the coupling of the terminal hydroxy group in vitamin-E, a typical Bio component to an ester linkage of a lysine methyl ester segment (typical link B component) within the BFSM""s central portion, requires the conversion of the ester to a carboxylic acid which can then undergo a condensation reaction with the hydroxyl of the vitamin-E molecule to yield the final BFSM. The latter coupling reaction can be further facilitated by incorporating better leaving groups than xe2x80x9cxe2x80x94OHxe2x80x9d on the carboxylic acid. Examples of preferred reagents include oxalyl chloride, ethylchlorofonnate, 1-ethyl-3-(3-dimethylamino-propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and N,Nxe2x80x2 carbonyldiimidazole. As well, it may be desired to have the drug extended out from the backbone of the BFSM to facilitate its biological role. This is typically done for anticoagulants like heparin, prior to their grafting onto substrates. Reaction of these particular components with a secondary carboxylic acid groups of the BFSM""s link B segments simply extends the secondary function of the BFSM for subsequent coupling of a drug molecule such as vitamin-E and GHG (the genetics housekeeping probe that is expressed in all cells to provide basic function needed for survival) procured from ACGT corp., Toronto ON
BFSMs can be synthesized with different components and stoichiometry. Prior to synthesis, the linkA and linkB molecules are, preferably, vacuum distilled to remove residual moisture. Bio compounds are dessicated to remove all moisture. Oligo components are degassed overnight to remove residual moisture and low molecular weight organics. Where BA-L is used as the fluoroalcohol, for example, this reagent is fractionated into three fractions to reduce the distribution of molecules with different xe2x80x9cmxe2x80x9d values. This reduces the selective reaction of a fluoro-alcohol of a particular xe2x80x9cmxe2x80x9d value over another, to provide for more control in the desired final product. The BA-L fractions are characterized as (i) a first fraction, herein called xe2x80x9cLxe2x80x9d fraction which is a clear liquid distilled at 102xc2x0 C. and atmospheric pressure; (ii) a second fraction referred to as xe2x80x9cIxe2x80x9d fraction, which is a white semi-solid material, distilled between 70 and 80xc2x0 C., under a vacuum of 0.01 mm Hg pressure; and (iii) a last fraction referred to as xe2x80x9cHxe2x80x9d fraction and is distilled between 80 and 100xc2x0 C., under a vacuum of 0.01 mm Hg and recovered as a very pale yellow solid. The selection of these fractions is somewhatarbitrary and it will be apparent to those skilled in the art that different fractions can be selected to alter the nature of the BFSM in order to tailor the material for specific applications with base polymers.
While reactants can be reacted in the absence of solvents if practical, it is preferable to use organic solvents compatible with the chemical nature of the reagents, in order to have good control over the characteristics of the final product. Typical organic solvents, include, for example, dimethylacetamide, acetone, tetrahydrofuran, ether, chloroform, dimethylsulfoxide and dimethylformamide. A preferred reaction solvent is N,N-dimethyleacetamide (DMAC, Aldrich Chemical Company, Milwaukee, Wis.).
In view of the low reaction activity of some diisocyanates, e.g. LDI and HDI, with oligo precursor diols, a catalyst is preferred for the synthesis. Typical catalysts are similar to those used in the synthesis of urethane chemistry and, include, dibutyltin dilaurate, stannous octoate, N,Nxe2x80x2 diethylcyclohexylamine, N-methylmorpholine, 1,4 diazo (2,2,2) bicyclo-octane and zirconium complexes such as Zr tetrakis (2,4-pentanedionato) complex.
In the first step of the preparation of a BFSM, for example, the linkB and linkA (optionally) reactant molecules are added to the oligo component and, optionally, catalyst to provide the xe2x80x9ccentral portionxe2x80x9d of the BFSM. Subsequently, the fluoro reactant component is added to the central portion and, generally, the mixture is allowed to react overnight. The product is precipitated in distilled water or a mixture of distilled water with methanol or ether. The latter step removes any residual fluoro reactant compound, while the product is dried under vacuum at 60xc2x0 C. Subsequent steps require activation of the secondary function on [linkB]. In a preferred case, this entails hydrolysis of a protective ester group on molecules such as LDI. This is achieved by dissolving the BFSM precursor in DMF and adjusting the acid content in the DMF solution, using an aqueous 1.0 N hydrochloric acid solution, to a pH reading of 1.5 on a pH meter. The solution temperature is then raised to 45xc2x0 C. and maintained at this temperature for 4 hours in order to permit the hydrolysis of the pendant ester groups. A base catalysed hydrolysis is also an option for de-esterification. The BFSM precursor is then precipitated in 1 M aqueous KCl, washed in distilled water and dried under vacuum at 60xc2x0 C. for 48 hours. The acid group of the BFSM precursor is then reacted with either oxalyl chloride, N,Nxe2x80x2 carbonyldiimidazole and 1-ethyl-3-(3-dimethylamino-propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) or other agents to introduce a good leaving group on the acid. This step permits for an efficient nucleophilic reaction to take place with the Bio component. In the preferred case oxalyl chloride is mixed with a solution of the acidified BFSM precursor in distilled DMF, in a nitrogen atmosphere. The solution is first cooled to 5xc2x0 C. with an ice bath and oxalyl chloride is added stoichiometrically to the amount of acid groups. Triethylamine is added to scavenge free HCl which is generated as a by-product. The latter reaction step produces an acid chloride BFSM precursor which is now ready to react with hydroxyl or amine groups on the Bio component. This could be the hydroxyl on vitamin-E, heparin or other oligosaccharides, hydrocortisone; the amine on norfloxacin, ciprofloxacin, phosphatidylcholine and phosphorylcholine derivatives, terminal amine of a peptide sequence, hydroxyl or amine of an oligonucleotide; or the amino/hydroxyl terminals of an ethylene oxide spacer group if so desired. The Bio molecule is dissolved into a suitable solvent, preferably DMF and added to the acid chloride BFSM precursor reaction mixture and the solution is allowed to react overnight at 20xc2x0 C. The final BFSM is precipitated in a mixture of ethanol/1 M aqueous KCl solution (30/70 vol %). The precipitated polymer is then washed three times in an 80/20 vol % ethanol/water mixture. Following washing, the material is dried under vacuum.
Fabrication of Product:
The BFSM""s are admixed with suitable amounts of base polymers in the fabrication of article products. The BFSM may be admixed with, for example, polyurethane base polymers by; 1) compounding methods for subsequent extrusion or injection molding or articles; 2) co-dissolving the polyurethane and BFSM into a solvent of common compatibility for subsequent casting of an article in a mold or for spinning fibers to fabricate an article; 3) wetting the surface of a polyurethane with a solution of BFSM in solvent of common compatibility with the polyurethane to which the BFSM solution is being applied; or 4) in admixture with a curable polyurethane, for example, 2 part curing system such as a veneer.
The invention, thus, provides in one aspect a series of novel polymeric additives, termed bioactive fluoroalkyl surface modifiers (BFSM) possessing intramolecular properties of biological activity endowed by distinct chemical functional groups and an affinity for polymeric surfaces endowed by polyfluoro oligomeric groups. When used in admixture with, for example, a polyurethane, the BFSMs establish a fluorocarbon base at the surface with a biological or pharmaceutical agent pendent adjacent to the fluorocarbon. The fluorocarbons provide a relatively neutral surface, in terms of surface energy, which does not promote strong cell adhesion, minimizes protein activation, and reduces biodegradation. This surface arrangement of fluorine chemistry is strategic, since in relation to the remainder of the surface, it permits the [Bio] component adjacent to fluorocarbon base to have a very specific cellular or biomolecular interaction with the intended target (i.e. platelet, bacteria, thrombin, etc.). When different BFSM""s are combined in admixture with the base polymer it permits for the development of a multifunctional surface, thus simultaneously addressing multiple issues of stability and bio-compatibility (e.g. coagulation, infection, inflammation, cell migration) related to implant materials. The BFSM""s are copolymers or terpolmers that have the ability to alter the surface chemistry and biochemistry and, hence, surface properties of a polymer and are synthesized in such a manner that (i) preferably, they have a lower molecular weight than the base material i.e. the polymer that requires the surface modification, (ii) they contain a surface active segment containing xcex1-xcfx89 terminal polyfluoro oligomeric groups and (iii) finally pendent drug or biologically active agents ([Bio]) that can be coupled to [linkB] components of the BFSM, for providing articles having bioactive surface properties, particularly for use in medical devices, promoting cell function and regulation, tissue integration, pro-active blood compatibility and specifically anti-coagulant/platelet function, biostability function, anti-microbial function and anti-inflammatory function, or for use in the biotechnology sector for improving affinity column chromatography systems or promoting surface catalytic reactions, or a biosensor and bio-diagnostic substrate.
Products such as medical devices formed of the admixed composition of the invention, have their surfaces modified as a result of the selective migration and interfacial localization of the low molecular weight oligomers containing pendent molecules with specific potential biological activity, carbon/fluorine segments and non-carbon/fluorine segments within the same molecule, such that the carbon/fluorine segments are terminal in the macromolecule and selectively reside at the material/environment interface, and such that the [Bio] moieties are coupled immediately adjacent, via the [linkB] segments, to the terminal carbon/fluorine segments of the macromolecule so that they also selectively reside at the surface of the material, while the non-carbon/fluorine segments are remote from the macromolecule""s terminal position, but reside within the upper surface of the product.
BFSMs, thus, contain, by synthesis through precursor-containing linkable-moieties such as hydroxyl, carboxylic acid and ester and preferably as pendent biological agents such as, for example, anti-inflammatory, agents such as, for example, non-steroidal-diflunisal via precursor hydroxyl, ibuprofen via carboxylic acid, naproxen via carboxylic acid, steroidal-hydrocortisone via hydroxyl, prednisolone via non-ring anti-coagulant agents, such as, heparin; anti-microbial agents, such as, fluoroquinolones such as norfloxacin, ciprofloxacin, sparfloxacin and trovafloxacin; and cell receptor ligands, such as, RGD integrin binding domain for a host of cells membranes, including macrophages, platelets, and the like, PHSRN (SEQ ID NO: 2), YRGDG (SEQ ID NO: 3), and RGDSPASSKP (SEQ ID NO: 4) amino acid sequences to promote cell activation and spreading; oligosaccharides, such as heparin sulfate, hyaluroic acid, dermitan sulfate, chondroitin 6-sulfate, keratan sulfate and heparin sulfate, for cell adhesion character in tissue engineering applications; oligonucleotides for binding DNA fragments and coupling of genes for bio-diagnostics, which this is best achieved with adapter sequences that have both strands of the cDNA that can protect the amine groups of the repeating nucleotides during coupling via the 5xe2x80x2 hydroxyl of the terminal nucleotide. Examples include aforesaid GHG and phospholipid head groups such as phosphatidylcholine and phosphorylcholine derivatives, for mimicking cell membranes. As well, BFSMs, thus, contain, preferably as xcex1-xcfx89 terminal polyfluoro oligomeric groups, fluoropolymeric segments comprising a sequential group of carbon atoms containing fluorine atoms and constituting an oligomeric chain. Preferred perfluorinated alcohols of use in the practice of the invention are those of the general formula CF3(CF2)nCH2CH2OH, having a linear alkyl chain, wherein n is 5-9, most preferably C8F17CH2CH2OH. These monomers are commercially available as homologous mixtures having varying degrees of fluoroalkane chain lengths. One such preferred mixture available under the name BA-L (Dupont trade marks-obtained from Van Waters and Rogers, Montreal, Canada) has an average molecular weight of 443; fluorine content of 70%; S.G. 1.5 @ 30xc2x0 C. thickening point  less than 25xc2x0 C. and a boiling range of 102-175xc2x0 C. @ 50 mm Hg.